This invention relates to methods and apparatuses for selecting specific cell types from cell suspensions, specifically those employing applied electric fields.
The ability to isolate specific sub-populations of cells from cell suspensions is of critical importance to many applications in the biological sciences as well as to many therapies in clinical medicine. For example, the basis of many medical therapies for treating a variety of human diseases and for countering the effects of a variety of physiological injuries involves the isolation, manipulation, expansion, and/or alteration of specific biological cells. One particularly important example involves the reconstitution of the hematopoietic system via bone marrow or progenitor cell transplantation. More specific examples include: autologous, syngeneic, and allogenic stem cell transplants for immune system reconstitution following the myeloablative effects of severe high dose chemotherapy or therapeutic irradiation; severe exposure to certain chemical agents; or severe exposure to environmental radiation, for example from nuclear weapons or accidents involving nuclear power generators.
Intensive chemotherapy and/or irradiation for the treatment of a variety of cancers, including breast cancer, has become a commonly used approach in cancer care centers. Such treatments are associated with severe ablation of the bone marrow cells required for function of the blood and immune systems. Such bone marrow cells are derived from a small number of progenitor cells known as hematopoietic stem cells in the normal bone marrow. Therefore, patients receiving such therapies require life-saving transplants of stem cells in order to survive the effects of the treatment. Stem cell containing tissue for transplant may be derived from donor marrow (allogeneic transplant) or from the patient""s own bone marrow or peripheral blood after mobilization (autologous transplant). In both instances, there is a need for effective cell separation methods to enrich the transplant tissue in stem cells and reduce the number of undesirable and deleterious cells (e.g. mature T cells for allogeneic transplants and residual cancerous cells for autologous transplants). For example, for autologous adjuvant stem cell transplant therapy following myeloablative cancer treatments, it is believed that reinfusion of residual tumor cells is a major cause of post therapy relapse. Clearly, removing such cells from transplanted tissue would be beneficial to the patient.
A number of cell isolation, cell separation, and cell purging strategies have been employed in the prior art for purifying or removing cells from a suspension. Prior art cell separation methods used to isolate cells or purge cell suspensions typically fall into one of three broad categories: physical separation methods typically exploit differences in a physical property between cell types, such as cell size or density (e.g. centrifugation or elutriation); chemical-based methods typically employ an agent that selectively kills or purges one or more undesirable cell types; and affinity-based methods typically exploit antibodies that bind selectively to marker molecules on a cell membrane surface of desired or undesired cell types, which antibodies may subsequently enable the cells to be isolated or removed from the suspension. While physical separation methods can be advantageous with regard to their ability to separate cells without causing undo damage to desired cells, current physical separation methods typically have relatively poor specificity and do not typically yield highly purified or highly purged cell suspensions. While many chemical and affinity methods have better selectivity than typical physical methods, they can often be expensive or time consuming to perform and can cause considerable damage to, or activation of, desired cells, for example stem cells, and/or can add undesirable agents to the purified or isolated cell suspensions (e.g. toxins, proliferation-inducing agents, and/or antibodies). An additional potential problem with antibody-based cell separation techniques typically employed for purification of stem cells, is that they select stem cells solely on the basis of cell surface markers (e.g., CD34) and will not select cells lacking such markers.
In addition to cancer therapy, there are a number of other important medical therapies which exist, or are under development, that are based on cells derived from a variety of different types of stem cells. Examples include pre-exposure prophylaxis or post-exposure therapies under development for a variety of biological exposures that may occur naturally (e.g., viral exposure for example with Ebola, etc.) or be inflicted by mankind (i.e., biological warfare agents). A variety of gene therapies involving genetically manipulated stem cells, are being contemplated or are under development for treating a variety of blood-related diseases (e.g., AIDS, leukemia, other cancers, etc.). Gene therapy techniques based on genetically manipulated stem and/or germ cells may also be useful in cloning organisms, such as animals. However, genetically manipulating stem cells using many current technologies is difficult, typically employing viruses or gene carriers that can be time consuming and expensive, or may be dangerous to perform and may not have high yields. Current research findings also suggest that the practical implementation of animal organ transplants into human recipients also may require procedures involving stem cells from both the donor and recipient. Many of these promising therapies would require cryopreservation and storage of donor specimens including human stem cells, for example, as derived from the stem cell-rich umbilical cord blood of newborns, which can provide such donors with a therapeutic basis for hematopoietic reconstitution or gene therapy should a health emergency occur later in life. If such storage demands are to be realistically met, the specimens will need to have minimal volume, and, therefore, successful implementation of such technologies may rest on the development and availability of effective methods for isolating trace numbers of stem cells from sources such as umbilical cord blood and the fetal liver. In order to achieve broad implementation of the therapies discussed above and others, rapid and cost effective methods are needed to isolate, with high purity, desired target cells from suspensions having a diverse mix of cell types and concentrations.
The use of applied electric fields to physically manipulate cells is known. Applied electric fields have been employed in the prior art for cell inactivation and sub-lethal cell membrane electroporation. For example, U.S. Pat. 5,048,404 to Bushnell discloses a system and method for sterilizing liquid foodstuffs by killing microorganisms with exposure to pulsed electric fields.
Sale and Hamilton (xe2x80x9cEffects of High Electric Fields on Microorganisms I. Killing of Bacteria and Yeasts,xe2x80x9d Biochim et Biophys Acta, 148:781 (1967); and xe2x80x9cEffects of High Electric Fields on Microorganisms II. Mechanism of Action of the Lethal Effect,xe2x80x9d Biochim et Biophys Acta, 148:789 (1967)) studied the effect of pulsed electric fields on suspensions of bacteria or suspensions of yeasts. Specifically, they investigated the effect on the degree of cell kill by the field as a function of field strength and exposure time. The effect of pulsed electric fields on the killing of bacteria was also studied by Hxc3xclsheger et al. (xe2x80x9cLethal Effects of High-Voltage Pulses on E. Coli K12,xe2x80x9d Radial Environ Biophys, 18:281(1980); and xe2x80x9cKilling of Bacteria with Electric Pulses of High Field Strength,xe2x80x9d Radial Environ Biophys, 20:53(1981)). Hxc3xclsheger et al. studied the effects on bacterial cell death of a variety of experimental parameters and were able to demonstrate a 99.9% reduction in the number of living bacterial cells in suspensions after exposure to certain pulsed electric field parameters.
The lysis of erythrocytes in erythrocyte suspensions by pulsed electric fields has also been studied both for bovine (Sale and Hamilton, xe2x80x9cEffects of High Electric Fields on Microorganisms III. Lysis of Erythrocytes and Protoplasts,xe2x80x9d Biochim et Biophys Acta, 163:37 (1967)) and human (Kinosita and Tsong, xe2x80x9cVoltage-Induced Pore Formation and Hemolysis of Human Erythrocytes,xe2x80x9d Biochim et Biophys Acta, 471:227 (1977); and Kinosita and Tsong, xe2x80x9cHemolysis of Human Erythrocytes by a Transient Electric Field,xe2x80x9d Proc Natl Acad Sci. 74:1923(1977)) erythrocytes. Knowledge derived from the studies above indicates that applied electric fields resulting in cellular transmembrane potentials on the order of 1 Volt can result in colloidal osmotic lysis of the erythrocytes.
Electric fields have also been used to sublethally porate the plasma membrane of nucleated cells, such as leukocytes and Chinese Hamster Ovary (CHO) cells (Sixou and Teissixc3xa9, xe2x80x9cSpecific Electropermeabilization of Leukocytes in a Blood Sample and Application to Large Volumes of Cells,xe2x80x9d Biochim et Biophys Acta, 1028:154 (1990)). Sixou and Teissixc3xa9 investigated electropermeabilization conditions to enable reversible poration of cell membranes, while maintaining long-term cell viability, for the purpose of enabling the reversibly porated cells to uptake drugs and act as immunocompatible drug delivery vehicles within the body. Sixou and Teissixc3xa9 studied the effect of pulsed electric field parameters on the reversible poration of suspensions comprising single cell types and suspensions comprising mixtures of two cell types (e.g. CHO cells and erythrocytes, and leukocytes and erythrocytes). The authors showed that reversible electropermeabilization is a function of the cell size and that large cells are reversibly porated at lower electric field strengths than small cells.
While the above mentioned methods and systems for cell separation and cell electropermeabilization represent, in some cases, valuable and useful techniques for some applications, there remains a need in the art for simple, fast, and clean methods to selectively isolate or remove specific cell sub-populations from cell suspensions without causing undo damage or activation to the remaining cells and without employing undesirable or toxic agents.
Accordingly, the present invention can provide relatively simple, fast, and clean methods for cell isolation or purging based on physical differences between different cell types present in a suspension. Furthermore, the invention provides systems and methods that enable selective isolation of viable cells, selective cell inactivation, as well as stem cell electropermeabilization, using applied electric fields.
In one aspect, the invention provides a method for creating from a biological sample having a given cell population, a suspension of cells that contain a selected viable subpopulation of the given cell population. The method is based on a characteristic electroporation threshold of the cells. The subpopulation of cells selected by the method is substantially limited to cells that have a characteristic electroporation threshold that is greater than a predetermined electroporation threshold. The selected suspension of cells is produced from the biological sample by first subjecting the sample to an electric field that has a magnitude that is sufficient to porate a substantial fraction of the cells in the sample that have a characteristic electroporation threshold less than the predetermined electroporation threshold. The electric field, however, does not porate a substantial fraction of cells that have a characteristic electroporation threshold greater than the predetermined electroporation threshold. Essentially, all of the porated cells in the sample that is subjected to the electric field are also inactivated.
In another aspect, the invention provides a method for creating a selected subpopulation of discreet objects from a sample having a given population of discreet objects. A discreet object comprises an inner conductive core which is surrounded by a dielectric membrane. The method is based on a characteristic electroporation threshold of the discrete objects. The subpopulation of discrete objects selected by the method is substantially limited to discrete objects that have a characteristic electroporation threshold that is greater than a predetermined electroporation threshold. The selected suspension of discrete objects is produced from the sample by first subjecting the sample to an electric field that has a magnitude that is sufficient to cause irreversible dielectric breakdown of the dielectric membrane of a substantial fraction of the discrete objects in the sample that have a characteristic electroporation threshold less than the predetermined electroporation threshold. The electric field, however, does not cause irreversible dielectric breakdown of the dielectric membrane of a substantial fraction of cells that have a characteristic electroporation threshold greater than the predetermined electroporation threshold.
In yet another aspect, the invention provides a method for porating cells. The method includes supplying a suspension of cells in a treatment volume, where the treatment volume includes at least two electrodes that are in fluid contact with the suspension. The method further involves applying a time varying bi-polar electrical potential across the electrodes that is sufficient to create an electric field that is sufficient to porate at least one cell in the suspension. The bi-polar electrical potential is varied so that the average current across the sample over the entire treatment time is essentially zero.
In another aspect, the invention provides the method for reversibly porating stem cells. The method involves supplying in a treatment volume a suspension of cells including a plurality of stem cells, which stem cells have a characteristic size, a characteristic shape, a plasma membrane, and a nuclear membrane. A pulsed electric field that has a pulse duration and magnitude sufficient to porate the plasma membrane of a cell having a characteristic size and shape essentially identical to the stem cells, but having an effective membrane thickness substantially exceeding the average membrane thickness of the plasma membrane of the stem cells is then applied to the suspension.
In another aspect, the invention involves a system for creating from a biological sample having a given cell population, a suspension containing a selected viable subpopulation of the given cell population. The selected cell population is substantially limited to cells that have a characteristic electroporation threshold greater than a predetermined electroporation threshold. The system functions by inactivating a substantial fraction of the cells in the sample not included in the selected subpopulation. The system includes a generating mechanism that generates an electric field of a magnitude and duration sufficient to irreversibly porate a substantial fraction of the cells not included in the selected subpopulation, while not irreversibly porating a substantial fraction of the cells included in the selected subpopulation. The system further includes a treatment cell that is electrically connected to the generating mechanism and is adapted to contain a cell suspension.
In yet another aspect, the invention provides a system for selectively inactivating biological cells based on a difference in a characteristic electric poration threshold. The system includes a generating mechanism that generates an electric signal constructed and arranged to create desired electric field parameters. The system also includes a treatment cell that is electrically connected to the generating mechanism, includes at least one electrode, and includes a treatment volume adapted to contain a cell suspension. The electrode is in fluid contact with the cell suspension during operation of the system and is constructed of a porous, biocompatible material, which is sealed in order to reduce the release of gases from the electrode during operation of the system.
In another aspect, the invention involves a cell suspension comprising a plurality of biological cells suspended in a liquid. The suspension includes one population of cells, which have a maximum of characteristic size not more than a predetermined value, that are substantially viable, and another population of cells, having a maximum characteristic size greater than the predetermined value, that are substantially non-viable. The cell suspension is obtained from a precursor suspension of substantially viable cells that contains as subpopulations the two cell populations mentioned above. The cell suspension is obtained by subjecting the precursor cell suspension to an electric field having a magnitude and duration that is sufficient to irreversibly porate a substantial fraction of the cells in the precursor suspension that have a maximum characteristic size above the predetermined value.
In yet another aspect, the invention involves a cell suspension comprising a plurality of biological cells suspended in a liquid where each of the biological cells is enclosed by a plasma membrane. The cell suspension includes a subpopulation of biological cells that possess a maximum characteristic size in excess of a predetermined value. Furthermore, the cells in the subpopulation of cells having a maximum characteristic size in excess of the predetermined, value also have a maximum transmembrane electrical potential that exceeds that required to cause irreversible dielectric breakdown of the plasma membrane of the cells.
In another aspect, the invention provides a cell suspension comprising a plurality of non-cultured biological cells, including a plurality of viable stem cells that have a given characteristic size, suspended in a liquid. The cell suspension further includes a plurality of irreversibly porated cells, essentially all of which irreversibly porated cells have a characteristic size that is greater than the characteristic size of the stem cells.
In another embodiment, the invention provides a cell suspension including a plurality of viable, reversibly electroporated stem cells.
In yet another aspect, the invention involves a suspension comprising viable, human pluripotent lympho-hematopoietic stem cells, which are capable of differentiating into members of the lymphoid, erythroid, and myeloid lineages. The suspension is essentially free of mature and lineage committed cells and is derived from a precursor cell suspension comprising substantially viable cells. The suspension is derived from the precursor suspension by subjecting the precursor suspension to an electric field of sufficient duration and magnitude to inactivate a substantial fraction of the mature and lineage committed cells in the precursor suspension.
Other advantages, novel features, and objects of the invention will be become apparent from the following detailed description of the invention when considered in conjunction with the accompanied drawings, which are schematic and which are not intended to be drawn to scale. In the figures, each identical or nearly identical component is illustrated in various figures is represented by a single numeral. For purposes of clarity, not every component is labeled in every figure.